Traditional processes of drug discovery involve the screening of complex fermentation broths and plant extracts for a desired biological activity or the chemical synthesis of many new compounds for evaluation as potential drugs. The advantage of screening mixtures from biological sources is that a large number of compounds are screened simultaneously, in some cases leading to the discovery of novel and complex natural products with activity that could not have been predicted otherwise. The disadvantages are that many different samples must be screened and numerous purifications must be carried out to identify the active component, often present only in trace amounts. On the other hand, laboratory syntheses give unambiguous products, but the preparation of each new structure requires significant amounts of resources. Generally, the de novo design of active compounds based on the high resolution structures of enzymes has not been successful.
In order to maximize the advantages of each classical approach, new strategies for combinatorial unrandomization have been developed independently by several groups. Selection techniques have been used with libraries of peptides (see Geysen, H. M., Rodda, S. J., Mason, T. J., Tribbick, G. & Schoofs, P. G., J. Immun. Meth. 1987, 102, 259-274; Houghten, R. A., Pinilla, C., Blondelle, S. E., Appel, J. R., Dooley, C. T. & Cuervo, J. H., Nature, 1991, 354, 84-86; Owens, R. A., Gesellchen, P. D., Houchins, B. J. & DiMarchi, R. D., Biochem. Biophys. Res. Commun., 1981, 181, 402-408), nucleic acids (see Wyatt, J. R., et al., Proc. Natl. Acad. Sci. USA, (in press); Ecker, D. J., Vickers, T. A., Hanecak, R., Driver, V. & Anderson, K., Nucleic Acids Res., 1993, 21, 1853-1856) and nonpeptides (see Simon, R. J., et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 9367-9371; Zuckermann, R. N., et al., J. Amer. Chem. Soc., 1992, 114, 10646-10647; Bartlett, Santi, Simon, PCT WO91/19735; and Ohlmeyer, M. H., et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 10922-10926). The techniques involve iterative synthesis and screening of increasingly simplified subsets of oligomers. Monomers or sub-monomers that have been utilized include amino acids and nucleotides both of which are bi-functional. Utilizing these techniques, libraries have been assayed for activity in either cell-based assays, or for binding or inhibition of purified protein targets.
A technique, called SURF (Synthetic Unrandomization of Randomized Fragments), involves the synthesis of subsets of oligomers containing a known residue at one fixed position and equimolar mixtures of residues at all other positions. For a library of oligomers four residues long containing three monomers (A, B, C), three subsets would be synthesized (NNAN, NNBN, NNCN, where N represents equal incorporation of each of the three monomers). Each subset is then screened in a functional assay and the best subset is identified (e.g. NNAN). A second set of libraries is synthesized and screened, each containing the fixed residue from the previous round, and a second fixed residue (e.g. ANAN, BNAN, CNAN). Through successive rounds of screening and synthesis, a unique sequence with activity in the assay can be identified. The SURF technique is described in Ecker, D. J., Vickers, T. A., Hanecak, R., Driver, V. & Anderson, K., Nucleic Acids Res., 1993, 21, 1853-1856. The SURF method is further described in PCT patent application WO 93/04204, the entire disclosure of which is herein incorporated by reference.
Phosphoramidates were note for use as protecting groups for ribooligonucleotide synthesis by Ohtsuka, E., et. al., Nucleic Acids Research, 1976, 3, 653. Oligonucleotides having a phosphoramidate link amino group at their 5' end were disclosed by Chu, B. C. F., et. al., Nucleic Acids Research, 1983, 11, 6513. Oligodeoxynucleotides (DNA) containing internucleotide phosphoramidate linkages have been synthesized by several groups. However, in each such synthesis, the phosphoramidate linkage has only been utilized to connect adjacent nucleosides, i.e. an internucleotide linkage. One of these synthesis was reported by Froehler, B., et. al., Nucleic Acids Research, 1988, 16, 4831-4838. As reported by Froehler, et. al., the stability of duplexes ranging from dimers to fifteen mers was studied to determine the ability of the oligonucleotides to hybridize to complementary diester oligonucleotides. Thermal denaturation revealed enhanced stability for dimers and trimers but less stability for longer sequences. Other phosphoramidite containing oligonucleotides are disclosed by Eritja, R. et. al., Tetrahedron, 1990, 45, 721; and Jager, A., et. al., Biochemistry, 1988, 27, 7237.
In U.S. Pat. No. 5,272,250, issued Dec. 21, 1993, boronated phosphoramidate compounds are disclosed. The disclosed compounds include a boron moiety connected through a tether to the nitrogen of the phosphoramidate. The boronated compound is present as either a monomer or linked to a nucleoside.
Modified oligodeoxynucleotides complementary to the RNA of human immunodeficiency virus 1 (HIV-1) were synthesized by Agrawal, S., et.al., Proc. Natl. Acad. Sci, USA., 1988, 85, 7079-7083). Among the modifications disclosed in this publication are phosphoramidate oligonucleotides. Groups attached to these phosphoramidates include butylamine, piperazidine, and morpholine. The interactions of these compounds to their target, i.e. RNA, was through an antisense mechanism utilizing normal Watson/Crick hydrogen bonding. Similar phosphoramidate containing oligonucleotides are further disclosed by Dagel, et. al., Nucleic Acids Research, 1990, 18, 4751.
Phosphoramidates oligonucleotides are also disclosed in European Patent Application 86307926.5, filed Oct. 14, 1986. In this patent, a number of functional groups are used for substituting onto the phosphoramidate nitrogen. As with others of the above referenced disclosures, again in this patent the groups linking the phosphoramidates are nucleosides of oligonucleotides.
A family of oligonucleotides of different lengths containing a cholesterol group or phenanthridinium group tethered via a phosphoramidate bond to an internucleoside phosphorous atom were synthesized and tested for activity in an HIV-1 assay by Letsinger, R. L., et.al., Proc. Natl. Acad. Sci. USA,. 1989, 86, 6553-6556). Two corresponding United States patents, U.S. Pat. Nos. 4,547,569 and 4,958,013, describes essentially the same structures.
In U.S. Pat. No. 5,218,103, issued Jun. 8, 1993, phosphorothioamidate oligonucleotides are disclosed. The phosphorothioamidate oligonucleotides disclosed in this patent are substituted with a variety of moieties on the phosphorothioamidate nitrogen.
In each of the foregoing disclosures, it is not known to use phosphoramidate linkage between any groups other than the nucleoside residues of oligonucleotides.